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MIST, a Novel Approach to Reveal Hidden Substrate Specificity in Aminoacyl-tRNA Synthetases.

Eriani G, Karam J, Jacinto J, Morris Richard E, Geslain R - PLoS ONE (2015)

Bottom Line: Although MIST is a non-cellular assay, it fully integrates the notion of tRNA competition.In particular, we demonstrate that yArgRS binds preferentially to type II tRNAs but does not support their misaminoacylation.Our results reveal important new trends in tRNA/AARS complex formation and potential deep physiological implications.

View Article: PubMed Central - PubMed

Affiliation: Architecture et Réactivité de l'ARN, Université de Strasbourg, CNRS, Institut de Biologie Moléculaire et Cellulaire, 67084, Strasbourg, CEDEX, France.

ABSTRACT
Aminoacyl-tRNA synthetases (AARSs) constitute a family of RNA-binding proteins, that participate in the translation of the genetic code, by covalently linking amino acids to appropriate tRNAs. Due to their fundamental importance for cell life, AARSs are likely to be one of the most ancient families of enzymes and have therefore been characterized extensively. Paradoxically, little is known about their capacity to discriminate tRNAs mainly because of the practical challenges that represent precise and systematic tRNA identification. This work describes a new technical and conceptual approach named MIST (Microarray Identification of Shifted tRNAs) designed to study the formation of tRNA/AARS complexes independently from the aminoacylation reaction. MIST combines electrophoretic mobility shift assays with microarray analyses. Although MIST is a non-cellular assay, it fully integrates the notion of tRNA competition. In this study we focus on yeast cytoplasmic Arginyl-tRNA synthetase (yArgRS) and investigate in depth its ability to discriminate cellular tRNAs. We report that yArgRS in submicromolar concentrations binds cognate and non-cognate tRNAs with a wide range of apparent affinities. In particular, we demonstrate that yArgRS binds preferentially to type II tRNAs but does not support their misaminoacylation. Our results reveal important new trends in tRNA/AARS complex formation and potential deep physiological implications.

No MeSH data available.


Related in: MedlinePlus

Northern blot analysis of yeast total RNAs under acidic conditions.Total RNAs were extracted and separated under acidic conditions before being transferred onto a nylon membrane. The membrane was probed with a mixture of 32P-labeled oligonucleotides complementary to tRNA Leu (UAA) and tRNA Arg (CCG). Acylated (+) and deacylated (-) tRNAs were loaded on separate lanes. The shift induced by the presence of arginine residues is significantly greater than the one induced by leucine. In vivo, tRNA Leu (UAA) is not significantly arginylated despite a strong affinity for yArgRS.
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pone.0130042.g007: Northern blot analysis of yeast total RNAs under acidic conditions.Total RNAs were extracted and separated under acidic conditions before being transferred onto a nylon membrane. The membrane was probed with a mixture of 32P-labeled oligonucleotides complementary to tRNA Leu (UAA) and tRNA Arg (CCG). Acylated (+) and deacylated (-) tRNAs were loaded on separate lanes. The shift induced by the presence of arginine residues is significantly greater than the one induced by leucine. In vivo, tRNA Leu (UAA) is not significantly arginylated despite a strong affinity for yArgRS.

Mentions: The gel mobility of aminoacyl-tRNAs depends both on the size and the charge of their amino acids. Leucine and arginine have comparable molecular weight (131 and 174 g/mol respectively) but different charges. With a pKa of 12.5, arginine is positively charged in acidic environments whereas leucine is electrically neutral. As a consequence, tRNA species simultaneously aminoacylated with arginine and leucine should yield two distinct bands by northern blot. As anticipated, we observed that the shift induced by arginine residues is significantly larger compared to leucine. On the northern blot, aminoacylated tRNA Leu migrate as a single spot demonstrating that misarginylation does not occur at a significant level (Fig 7). Despite a superior binding ability, non-cognate tRNA Leu (UAA) are not significantly aminoacylated by yArgRS in vivo.


MIST, a Novel Approach to Reveal Hidden Substrate Specificity in Aminoacyl-tRNA Synthetases.

Eriani G, Karam J, Jacinto J, Morris Richard E, Geslain R - PLoS ONE (2015)

Northern blot analysis of yeast total RNAs under acidic conditions.Total RNAs were extracted and separated under acidic conditions before being transferred onto a nylon membrane. The membrane was probed with a mixture of 32P-labeled oligonucleotides complementary to tRNA Leu (UAA) and tRNA Arg (CCG). Acylated (+) and deacylated (-) tRNAs were loaded on separate lanes. The shift induced by the presence of arginine residues is significantly greater than the one induced by leucine. In vivo, tRNA Leu (UAA) is not significantly arginylated despite a strong affinity for yArgRS.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4465971&req=5

pone.0130042.g007: Northern blot analysis of yeast total RNAs under acidic conditions.Total RNAs were extracted and separated under acidic conditions before being transferred onto a nylon membrane. The membrane was probed with a mixture of 32P-labeled oligonucleotides complementary to tRNA Leu (UAA) and tRNA Arg (CCG). Acylated (+) and deacylated (-) tRNAs were loaded on separate lanes. The shift induced by the presence of arginine residues is significantly greater than the one induced by leucine. In vivo, tRNA Leu (UAA) is not significantly arginylated despite a strong affinity for yArgRS.
Mentions: The gel mobility of aminoacyl-tRNAs depends both on the size and the charge of their amino acids. Leucine and arginine have comparable molecular weight (131 and 174 g/mol respectively) but different charges. With a pKa of 12.5, arginine is positively charged in acidic environments whereas leucine is electrically neutral. As a consequence, tRNA species simultaneously aminoacylated with arginine and leucine should yield two distinct bands by northern blot. As anticipated, we observed that the shift induced by arginine residues is significantly larger compared to leucine. On the northern blot, aminoacylated tRNA Leu migrate as a single spot demonstrating that misarginylation does not occur at a significant level (Fig 7). Despite a superior binding ability, non-cognate tRNA Leu (UAA) are not significantly aminoacylated by yArgRS in vivo.

Bottom Line: Although MIST is a non-cellular assay, it fully integrates the notion of tRNA competition.In particular, we demonstrate that yArgRS binds preferentially to type II tRNAs but does not support their misaminoacylation.Our results reveal important new trends in tRNA/AARS complex formation and potential deep physiological implications.

View Article: PubMed Central - PubMed

Affiliation: Architecture et Réactivité de l'ARN, Université de Strasbourg, CNRS, Institut de Biologie Moléculaire et Cellulaire, 67084, Strasbourg, CEDEX, France.

ABSTRACT
Aminoacyl-tRNA synthetases (AARSs) constitute a family of RNA-binding proteins, that participate in the translation of the genetic code, by covalently linking amino acids to appropriate tRNAs. Due to their fundamental importance for cell life, AARSs are likely to be one of the most ancient families of enzymes and have therefore been characterized extensively. Paradoxically, little is known about their capacity to discriminate tRNAs mainly because of the practical challenges that represent precise and systematic tRNA identification. This work describes a new technical and conceptual approach named MIST (Microarray Identification of Shifted tRNAs) designed to study the formation of tRNA/AARS complexes independently from the aminoacylation reaction. MIST combines electrophoretic mobility shift assays with microarray analyses. Although MIST is a non-cellular assay, it fully integrates the notion of tRNA competition. In this study we focus on yeast cytoplasmic Arginyl-tRNA synthetase (yArgRS) and investigate in depth its ability to discriminate cellular tRNAs. We report that yArgRS in submicromolar concentrations binds cognate and non-cognate tRNAs with a wide range of apparent affinities. In particular, we demonstrate that yArgRS binds preferentially to type II tRNAs but does not support their misaminoacylation. Our results reveal important new trends in tRNA/AARS complex formation and potential deep physiological implications.

No MeSH data available.


Related in: MedlinePlus